DIRECTING POTENTIAL ANTI-XYLELLA GENE PRODUCTS TO THE XYLEM OF TRANSGENIC GRAPEVINES Project Leaders:

The purpose of this research was to transform Vitis vinifera cultivars with the pear polygalacturonase inhibiting protein (PGIP) gene in order to analyze its effect in developing resistance to PD in transgenic plants. A second goal was the transformation of grapevine with several green fluorescence protein (GFP) constructs carrying sequences expected to enhance secretion from the cell to evaluate the effect of signal sequences on the targeting of transgene products to xylem tissue. Some of the transgenic lines expressing pgip exhibited reduced PD symptoms, which suggests that Xyllela polygalaturonase might be inhibited in transgenic plants. Tests will be conducted in the future to evaluate the development of PD in the field. We also found that the pear PGIP was secreted into the xylem. This is relevant to PD because X. fastidiosa is a xylem-limited bacteria. It is also very important that the transgene product was observed to move through the graft union and thus is transmitted to the scion, implying that a few transgenic rootstocks could be used with any scion variety. Fluorescence in plants transformed with GFP fused to the signal peptide sequences of tricosanthin and XSP30 was only detected inside the cells. The absence of fluorescence in the apoplast could be related to GFP expression itself instead of failure of TCS and XSP30 signal peptides. INTRODUCTION Genetic engineering offers the possibility of introducing genes that will improve tolerance to Pierce’s disease in existing grape varieties without otherwise changing their viticultural or enological characteristics. One of our targets is a gene coding for a pear PGIP cloned in the Labavitch lab (Stotz et al. 1993). PGIP’s are plant cell wall proteins that specifically inhibit fungal polygalacturonases (PG). By inhibiting PGs, PGIP’s interfere directly with host cell wall degradation and may prevent degradation of pectic oligomeric elicitors that are inducers of the plant defense response. Their role in plant defense suggests that they may be useful for engineering transgenic plants resistant to pathogen infection. Powell et al. (2000) showed that transgenic tomato plants transformed with the pear PGIP gene exhibited reduced susceptibility to infection with Botrytis cinerea. The fact that Xylella fastidiosa, the causal agent of PD in grapevines, has genes putatively encoding PG and other cell wall-degrading enzymes (Simpson et al., 2000) led us to hypothesize that PGIP could confer tolerance against Xylella in grapes. In order to test this hypothesis, pre-embryogenic calluses originating from anthers of Vitis vinifera cvs. ‘Thompson Seedless’ and ‘Chardonnay’ were cultivated with Agrobacterium tumefaciens harboring binary plasmid pDU94.0928, that contains the pear PGIP gene under the control of the CaMV 35S promoter. We are also investigating the targeting of transgene products to xylem tissue. Because X. fastidiosa is xylem limited, it will be essential that any anti-Xylella gene product be present in the xylem in an effective concentration. In order to study protein secretion in grape, pre-embryogenic calli originating from anthers of Vitis vinifera cvs. ‘Thompson Seedless’ and ‘Chardonnay’ were cultivated with Agrobacterium tumefaciens carrying three gene constructs that included the coding sequence for a synthetic GFP (Maximova et al 1998) and GFP fused with amino-terminal of the secreted protein tricosanthin (TCS) (Krishnan 2000) or the xylem specific protein XSP30 (Masuda et al, 1999), all under the control of the Ca MV 35S promoter. OBJECTIVES 1. Characterize the role of the transgene in the delayed development of PD in transgenic grapevines that express the pear PGIP. 2. Measure the abundance of marker gene product in the xylem sap of transgenic plants and non-transgenic scions grafted into transgenic rootstocks. 3. Evaluate the effect of signal sequences on the targeting of transgene products to xylem tissue.